The present invention relates to optical tomography systems in general, and more particularly to those tomography systems that utilize an illumination system comprised of photon beams wherein each beam consists of photons that are not parallel, such as a cone beam or a fan beam for illuminating fluorescent molecular probes.
The use of molecular probes in the analysis of biological cells has illuminated many aspects of the disease process in cells. Molecular probes are used in several applications, and those tagged with fluorescent molecules have been particularly useful. However, it has not been possible to combine highly quantitative measurements of the amount of bound probes within a cell with the precise three-dimensional (3D) localization of these probes within subcellular structures.
U.S. application Ser. No. 09/927,151 of Alan C. Nelson, filed Aug. 10, 2001, entitled xe2x80x9cAPPARATUS AND METHOD FOR IMAGING SMALL OBJECTS IN A FLOW STREAM USING OPTICAL TOMOGRAPHY,xe2x80x9d and U.S. application Ser. No. 10/126,026 of Alan C. Nelson, filed Aug. 19, 2002, entitled xe2x80x9cVARIABLE-MOTION OPTICAL TOMOGRAPHY OF SMALL OBJECTSxe2x80x9d (hereinafter called the FOT and VOT systems, respectively, or the optical tomography systems (OT systems), taken together) are both incorporated herein by this reference. In the aforesaid Nelson patent applications, OT systems are described that utilize divergent optical beams, such as a cone beam or a fan beam, in geometries that facilitate the 3D computed image reconstruction of small objects such as biological cells. As input to reconstruction algorithms, these OT systems generate a set of projection images, also called shadowgrams that are generated by detecting absorption of photons from the primary beam by an object of interest. Unfortunately, the OT systems do not address the additional case where molecules within the object of interest emit secondary photons when illuminated by the primary beam, and hence, a new methodology is required to compute the 3D arrangement of these probes through emission computed tomography.
Localizing sources of emission from within an object of interest, a cell for example, has been a significant challenge to the scientific community. Those skilled in the art will recognize that even current solutions are inadequate, including the use of collimation at the detector to determine the solid angle direction of detected photons and the use of precise time-of-flight measurements to track the distance from the detector to the emission source.
In one embodiment, the present invention provides a method for emission computed tomography (ECT). In accordance with one example of the present invention, the relative motion of an object of interest, such as a cell, through a non-parallel beam, such as a cone beam or a fan beam, where the cell has been exposed to molecular probes that can fluoresce while passing through the beam, generates temporal signatures. The temporal signatures are combined with a tomographic reconstruction from projections to generate an accurate 3D image of the fluorescent tagged subcellular structures. Using the temporal signatures of objects tagged with fluorescent molecules moving relative to such beams in conjunction with tomographic reconstruction from projections, the fluorescent signals are accurately measured and localized.
In the case of tomographic image reconstruction from projections, the present invention overcomes the drawbacks of the prior art by, for the first time, advantageously using the reconstructed image in conjunction with measurements of emission from within the cell to pinpoint the location of internal emission sources. One example of the method of the invention employs non-parallel primary beams, such as cone beams or fan beams, which are divergent beams, coupled with relative motion between the beam and the cell to generate a set of temporal signatures that are mapped into the cell through a combination of hyperbolic surfaces and pattern matching to uniquely localize the emission sources within the reconstructed cell image.